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Abstract

Introduction

In critically ill patients, the appearance of nucleated red blood cells (NRBCs) in
blood is associated with a variety of severe diseases. Generally, when NRBCs are detected
in the patients' blood, the prognosis is poor.

Methods

In a prospective study, the detection of NRBCs was used for a daily monitoring of
383 medical intensive care patients.

Results

The incidence of NRBCs in medical intensive care patients was 17.5% (67/383). The
mortality of NRBC-positive patients was 50.7% (34/67); this was significantly higher
(p < 0.001) than the mortality of NRBC-negative patients (9.8%, 31/316). Mortality increased
with increasing NRBC concentration. Seventy-eight point six percent of the patients
with NRBCs of more than 200/μl died. The detection of NRBCs is highly predictive of
death, the odds ratio after adjustment for other laboratory and clinical prognostic
indicators being 1.987 (p < 0.01) for each increase in the NRBC category (0/μl, 1 to 100/μl, 101 to 200/μl,
and more than 200/μl). Each step-up in the NRBC category increased the mortality risk
as much as an increase in APACHE II (Acute Physiology and Chronic Health Evaluation
II) score of approximately 4 points. The mortality of patients who were NRBC-positive
on the day of relocation from the intensive care unit to a peripheral ward was 27.6%
(8/27). This was significantly higher than the mortality of patients who were NRBC-negative
on the relocation day (8.6%, 28/325; p < 0.01). On average, NRBCs were detected for the first time 14 days (median, 3 days)
before death.

Conclusion

The routine analysis of NRBCs in blood is of high prognostic power with regard to
mortality of critically ill patients. Therefore, this parameter may serve as a daily
indicator of patients at high mortality risk. Furthermore, NRBC-positive intensive
care patients should not be relocated to a normal ward but should obtain ongoing intensive
care treatment.

Introduction

Under normal conditions, the peripheral blood of healthy adults is generally free
of nucleated red blood cells (NRBCs), which tend to be found in patients with severe
diseases [1-5] who have a relatively poor prognosis [3,4,6-9]. In most of the earlier studies on NRBCs, the concentration was determined microscopically
by a stained peripheral blood smear. With such a technique, it is difficult to detect
NRBC concentrations of less than 200/μl [10]. For several years, a more convenient and sensitive technique has been available
in the form of mechanized blood analyzers. With such an analyzer, one can routinely
determine NRBC concentrations of less than 100/μl [11-13] The results of our recent studies with this new technique indicate that the detection
of NRBCs may serve as an early indicator in patients at increased risk of mortality:
on average, the presence of NRBCs was detected 1 to 3 weeks before death [14,15]. Furthermore, the analysis of the cytokine profile in the blood of NRBC-positive
patients (without hematologic diseases) suggests that NRBCs may be considered a parameter
that sums hypoxic and inflammatory injuries. This may be the reason why the appearance
of NRBCs is a strong predictor of increased mortality [15-17].

Recently, we reported on the poor prognosis of surgical intensive care patients when
NRBCs are found in the peripheral blood [18]. In that study, for the first time, a systematic day-to-day screening for NRBCs in
the blood of surgical intensive care patients was performed. Our study revealed that
32% of the surgical intensive care patients were NRBC-positive at least once. The
detection of NRBCs was associated with a greatly increased mortality of 44% (versus
4% of NRBC-negative patients). The area under the curve amounted to 0.86. On average,
NRBCs were detected nine days before death. Therefore, in the present study, we set
out to establish whether the daily screening for NRBCs in medical intensive care patients
could serve as an early indicator of medical intensive care patients at extremely
high risk.

Our study revealed that 18% of the medical intensive care patients were NRBC-positive.
The detection of NRBCs was associated with a greatly increased in-hospital mortality.
More than 50% of the NRBC-positive patients died. Furthermore, the mortality was three
times higher in patients who were NRBC-positive on the day of relocation from the
intensive care unit to a peripheral ward compared to patients who were NRBC-negative
on the relocation day. On average, NRBCs were detected 14 days before death. These
results suggest that the routine daily measurement of NRBCs could aid in a daily risk
assessment of medical intensive care patients.

Materials and methods

Subjects and protocol

All intensive care patients treated between April 2003 and January 2004 in the intensive
care unit of the Department of Internal Medicine of Berufsgenossenschaftliche Universitaetsklinik
Bergmannsheil GmbH (University Hospital, Ruhr-University Bochum, Germany) (n = 383) were included in this study. Patients younger than 18 years and patients after
surgery were excluded from this study. To evaluate the prognostic significance of
NRBCs in the peripheral blood of medical intensive care patients, we screened one
blood sample of each patient each day by means of a Sysmex XE-2100 (Sysmex Europe
GmbH, Norderstedt, Germany). Blood samples were routinely drawn in the morning. For
statistical analysis, a patient was defined as NRBC-positive when NRBCs were detected
in the blood at least once. Outcome was considered as in-hospital mortality. Ethical
approval to conduct this study was granted by the Ethical Committee of Ruhr-University
Bochum (reference no. 1982).

Laboratory tests

Blood count parameters (NRBCs, leukocytes, hemoglobin, and thrombocytes) were measured
using a Sysmex XE-2100 blood analyzer in line with the manufacturer's recommendations.
According to the manufacturer, the NRBC detection limit was greater than 19/μl. Stringent
internal quality control measurements were performed, and the criteria of acceptance
were fulfilled throughout.

Creatinine, alanine aminotransferase, and C-reactive protein were measured with an
LX 20 analyzer (Beckman Coulter GmbH, Krefeld, Germany), and prothrombine time ratio
was assayed with a BCS (Behring Coagulation System) (Dade Behring, Schwalbach, Germany),
all in accordance with the recommendations of the manufacturers. The quality assurance
of quantitative determinations was strictly performed according to the German Norm:
Quality Assurance in Medical Laboratories (DIN [Deutsches Institut für Normung] 58936,
2000). The criteria of acceptance were fulfilled throughout. Retrospective analysis
of the laboratory data revealed 0.3% missing values.

Statistical analysis

Data are presented as the mean ± standard error of mean. When samples were normally
distributed, the differences between the data for survivors and deceased were analyzed
using the t test procedure. When samples were not normally distributed, the Mann-Whitney test
was used because this test does not require a normal distribution of data. In the
case of categorical data, the Fisher exact test was used. Correlations were analyzed
by Pearson or non-parametric Spearman correlation. A p value of less than 0.05 was considered statistically significant.

A receiver operating characteristic curve was obtained by plotting the true-positive
proportion (sensitivity) against the false-negative proportion (1 – specificity).
The area under the curve (C-statistics) was calculated by non-linear regression.

The prognostic significance of NRBCs and other risk indicators was assessed using
multiple logistic regression. In this study, the logistic regression tries to estimate
the relative effect that parameters have on the patients' outcome. This is facilitated
by assuming a functional relationship (the 'logistic model') between variables and
probability of outcome. Then, for all possible settings, every variable is given a
relative importance that makes the actual observed event 'most likely', taking into
account the effects of all other variables. This is called the 'maximum likelihood'
estimate of the variables' influence. These coefficients provide a relative weighting
for each variable. Moreover, they can be used to derive odds ratios for the variables.
If the odds ratio differs significantly from 1, a significant prognostic power that
is independent of the other variables considered may be assumed.

In a first step, laboratory data were analyzed with regard to mortality. If reasonable
for calculation of the odds ratios, the data were categorized in up to four categories.
That is, NRBCs were subdivided into four categories: 0/μl, 1 to 100/μl, 101 to 200/μl,
and more than 200/μl. A backward selection multiple logistic regression analysis was
performed by first including all parameters in a multivariate model and subsequently
leaving out the parameters with the largest p values until no parameter with a p value greater than 0.25 was included. The calculations were carried out using SAS
version 8.02 (SAS Institute Inc., Cary, NC, USA). The intention of this study was
to evaluate the prognostic power of the presence of NRBCs in the blood with regard
to the patients' in-hospital mortality risk.

Results

Patient characteristics

We included 383 medical intensive care patients. The mean age was 66.3 ± 0.8 years
(range, 20 to 94 years). Two hundred twenty-five male (58.7%) and 158 female (41.3%)
patients were included in this study. On average, patients were treated for 4.1 ±
0.3 days (n = 383) in the intensive care unit. Total mortality was 17.0% (65/383). The APACHE
II score and the SAPS II amounted to 16.0 ± 0.5 and 35.2 ± 0.9, respectively.

The incidence of NRBCs in the blood was 17.5% (67/383). No significant difference
was found between the incidences in male (16.4%, 37/225) and female (19.0%, 30/158)
patients. On the day of admission, 7.8% (30/383) of the patients were NRBC-positive.
On average, NRBCs were detected for the first time on the third day of intensive care
treatment (3.1 ± 0.4; Figure 1), but in 44.8% (30/67) of the NRBC-positive patients, NRBCs were already detected
on the admission day. On average, the highest NRBC concentration of each individual
NRBC-positive patient was 189 ± 41/μl (range, 20 to 1,760/μl; median, 80/μl; n = 67). Some of the basal clinical characteristics of NRBC-positive and NRBC-negative
patients are summarized in Table 1.

Figure 1. Intensive care days on which nucleated red blood cells were detected for the first
time in the blood of medical intensive care patients.

Prognostic significance of nucleated red blood cells

The mortality of NRBC-positive patients was 50.7% (34/67). The predictive value for
death increased with higher NRBC concentrations (Figure 2). The mortality was 46.7% (14/30) in patients who were NRBC-positive on the day of
admission to the intensive care unit. In contrast, the mortality of NRBC-negative
patients was 9.8% (31/316; p < 0.001).

Figure 2. In-hospital mortality of medical intensive care patients in relation to the concentration
of nucleated red blood cells (NRBCs) in the blood. Numbers in parenthesis denote the
ratio of deceased patients to all patients with the respective NRBC concentration.

Furthermore, the mortality of patients who were NRBC-positive on the day of relocation
from the intensive care unit to a peripheral ward was 27.6% (8/27). This was significantly
higher than the mortality of patients who were NRBC-negative on the relocation day
(8.6%, 28/325; p < 0.01).

Overall, with regard to in-hospital mortality, NRBCs in blood showed sensitivity and
specificity of 52.3% and 89.6%, respectively. The area under the curve was 0.72.

NRBCs were an early indicator of patients at increased mortality risk. On average,
in NRBC-positive patients who died, NRBCs were detected for the first time 13.6 ±
3.8 days (median, 3 days; n = 34) before death.

After the first detection of NRBCs in blood and during the further course of intensive
care treatment, when the NRBCs have disappeared from the circulation, the mortality
again decreased. That is, when former NRBC-positive patients were again NRBC-negative
for more than 4 days after the final detection of NRBCs in blood, the mortality decreased
to 16.7% (1/6).

As shown in Figure 3, the appearance of NRBCs in blood seems not to be associated with one particular
cause of death. However, patients who have died from infections or sepsis, in particular,
had significantly higher NRBC concentrations than patients who have died from cerebral
or pulmonary complications. None of the other defined causes of death was associated
with an NRBC concentration that was significantly higher than the others.

Figure 3. Concentration of nucleated red blood cells (NRBCs) in the blood of medical intensive
care patients who have died from various causes. ◆ indicate the NRBC concentration
of each individual deceased patient. The average concentration is indicated by horizontal
bars. denote the significance of the difference.

Nucleated red blood cells in relation to other clinical and laboratory risk indicators

Table 2. Incidence of NRBCs in blood in medical intensive care patients in relation to the
APACHE II and the SAPS II

The Spearman correlation of the NRBCs with other laboratory parameters is displayed
in Table 3. When correlation was calculated with values measured on the day of the first appearance
of NRBCs in blood, NRBCs significantly increased with the leukocytes (r = 0.373, p < 0.01) and the creatinine concentration (r = 0.284, p < 0.05). Moreover, NRBCs increased with a decreasing prothrombin time ratio (r = -0.408, p < 0.001). The concentrations of hemoglobin, thrombocytes, and C-reactive protein as
well as the alanine aminotransferase activity were not significantly correlated with
the NRBC concentration.

The detection of NRBCs was an independent risk indicator of poor outcome. In terms
of mortality, the odds ratio for each stepwise increase in the NRBC categories was
calculated in relation to other clinical and laboratory risk indicators by means of
multiple logistic regression (Table 4). Because the correlation coefficient calculated by linear regression between APACHE
II score and SAPS II was r = 0.91, only the APACHE II score was considered for the multiple logistic regression.
The detection of NRBCs is highly predictive of death, the odds ratio after adjustment
for other clinical and laboratory prognostic indicators being 1.987 (p < 0.01) for each increase in the NRBC category (0/μl, 1 to 100/μl, 101 to 200/μl,
and more than 200/μl). That is, patients with more than 200/μl NRBCs had a more than
seven-fold higher risk of dying than patients without NRBCs.

Furthermore, under consideration of the APACHE II score, the 'maximum likelihood estimate'
for each increase in the NRBC category was 0.687 ± 0.253 and therefore approximately
four times (exactly 4.40 times) higher than the 'maximum likelihood estimate' for
each one-point increase in the APACHE II score (0.156 ± 0.025). Therefore, each step-up
in the NRBC category is equivalent to approximately 4 APACHE II score points. Consequently,
an adjustment of the APACHE II score could be performed by adding those 4, 8, or 12
points dependent on the patient's NRBC category (1 to 100, 101 to 200, and more than
200/μl) to the individual APACHE II score of NRBC-positive patients. In practice,
this modification would be a reassessment of the patient's prognosis. The area under
the curve for this modified APACHE II score was 0.91 compared to 0.87 and 0.72 for
the APACHE II score and the NRBCs alone, respectively (Table 5).

Discussion

To our knowledge, this is the first study in which the detection of NRBCs in the peripheral
blood was investigated with regard to its prognostic significance for the intensive
care mortality of medical intensive care patients. In earlier studies, we and others
have shown that the detection of NRBCs is associated with a relatively poor prognosis
[3,4,6-8,10,14,21,22]. In most of those studies, the NRBC detection and quantification were based on the
microscopic analysis of stained blood smears. This technique is time-consuming and
only partly suitable for the detection and quantification of NRBC concentrations of
less than 200/μl [10].

In this study, the NRBC concentration was screened with a mechanized blood analyzer
of high sensitivity [11,12,23]. The present study revealed that approximately 18% of all medical intensive care
patients were NRBC-positive at least once. Interestingly, in nearly half of the NRBC-positive
patients, NRBCs were detected already on the admission day.

Our data confirmed the high prognostic power of the mechanized detection of NRBCs
in blood in terms of mortality. The total in-hospital mortality of NRBC-positive patients
of this study was 50.7%. Furthermore, as shown in earlier studies, the present data
showed that the mortality increased with an increasing NRBC concentration [10,24,25] Approximately 80% of the patients with NRBC concentrations higher than 200/μl died.

Our study revealed that the daily screening for NRBCs can be used to estimate the
patients' mortality risk. Not only did the predictive value for death increase with
the concentration of NRBCs in the blood, but the prognosis improved when the NRBC
concentration decreased. In particular, after the first detection of NRBCs in blood
and during the further course of intensive care treatment, when the NRBCs have disappeared
from the circulation for more than four days, the mortality again significantly decreased
nearly to values of NRBC-negative patients [18].

In the present study, increased creatinine and leukocyte concentrations and a lower
prothrombin time ratio were significantly correlated with increased NRBC concentrations.
Although these findings suggest that NRBC-positive patients are more severely burdened
than NRBC-negative patients, the detection of NRBCs is an independent predictor of
poor outcome. To evaluate the independent attributable risk factor, a logistic regression
considering NRBCs, age, gender, body mass index, APACHE II score, creatinine, hemoglobin,
thrombocytes, leukocyte, alanine aminotransferase, C-reactive protein, and the prothrombin
time ratio was performed. As a result, the independent prognostic power of NRBCs is
underlined by an odds ratio of 1.987 for each stepwise increase in the NRBC category.
That is, patients with NRBCs of more than 200/μl have a more than seven-fold higher
risk to die than NRBC-negative patients. In recent studies, we have already demonstrated
that the detection of NRBCs is a risk indicator that is independent of several other
established risk indicators [10,14,16,24].

Among the general severity of illness scoring systems for intensive care patients,
APACHE II and SAPS II have become two of the most accepted and used [26-30]. However, the present data suggest that the APACHE II score could be significantly
improved by adding up to 12 score points, considering the presence of NRBCs as an
independent variable in this score, as suggested for the abbreviated burn severity
index in patients with burns [25].

The analysis of the lifespan of the patients who died indicates that NRBCs in blood
were found not just immediately before death. Moreover, our present study showed that
the detection of NRBCs is often a relatively early phenomenon prior to death. In deceased
patients, NRBCs were detected 14 days before death. Therefore, NRBCs would seem to
be an early indicator of increased risk.

Finally, the underlying pathophysiology of NRBCs in blood is not fully understood.
In our study, no association with only one of the various causes of patient death
was found. However, some authors have claimed that hypoxemia [31,32], acute and chronic anemia [33,34], or severe infections [35,36] are linked to the appearance of NRBCs in critically ill patients. In this context,
we recently reported on the cytokine profile and the erythropoietin concentrations
in NRBC-positive patients [17]. Our data suggested an important role of inflammation and/or decreased tissue oxygenation
(caused by local or systemic circulatory disorders) for the appearance of NRBCs in
blood. NRBCs may thus be considered a marker that sums up hypoxic and inflammatory
injuries. It seems obvious that these complications have an impact on patient prognosis.
Therefore, this could be the reason why the appearance of NRBCs is a strong predictor
of increased mortality.

However, concerning such an association between inflammation (with or without hypoxia)
and NRBCs, it is attractive to speculate what kind of therapy could improve the poor
prognosis of NRBC-positive patients. Currently, studies are under way in our university
hospital to show whether intensifying the treatment of patients with NRBCs (that is,
an earlier administration of antibiotics or an anti-inflammatory therapy) can reduce
their mortality rate.

Nonetheless, we observed that the mortality was three times higher in patients who
were NRBC-positive on the day of relocation from the intensive care unit to a normal
ward compared to patients who were NRBC-negative on the relocation day. Consequently,
it seems obvious that NRBC-positive patients should obtain ongoing intensive care
treatment.

Conclusion

This is the first study in which the daily screening for NRBCs in the peripheral blood
of patients in the medical intensive care unit was investigated with regard to its
prognostic power for in-hospital mortality. The incidence of NRBC-positive patients
was 18%. NRBC detection in critically ill patients was associated with significantly
increased in-hospital mortality (50.7% versus 9.8%). The predictive value for death
increased with the NRBC concentration and seems to decline again when the NRBCs have
disappeared from the circulation. The prognostic significance of NRBCs was independent
of other laboratory and clinical risk parameters. An improvement of established risk
models like APACHE II seems feasible. Furthermore, the detection of NRBCs in blood
is a relatively early phenomenon prior to death, so screening for NRBCs may aid in
the early identification of patients at high risk. Further studies are needed to clarify
whether the detection of NRBCs could help to decide on a change of patient management,
but our present data suggest that NRBC-positive patients should obtain ongoing intensive
care treatment.

Key messages

• The detection of NRBCs in the blood of medical intensive care patients is associated
with significantly increased in-hospital mortality (50.7% versus 9.8%). The prognostic
significance of NRBCs was independent of other laboratory and clinical risk parameters.
The predictive value for death increased with the NRBC concentration and seems to
decline again when the NRBCs have disappeared from the circulation.

Abbreviations

Competing interests

AS and RK obtained a grant from Sysmex Europe GmbH (Norderstedt, Germany) to perform
this study. The other authors declare that they have no competing interests.

Authors' contributions

All authors made substantive intellectual contributions to the design and conception
of this study. AS was responsible for data acquisition and data presentation, performed
the analysis and interpretation of data, and was responsible for the writing of the
manuscript. RK and ES were responsible for data acquisition and data presentation
and performed the analysis and interpretation of data. SH was responsible for data
acquisition and data presentation. TH-L performed the analysis and interpretation
of data. MK was responsible for the writing of the manuscript. All authors read and
approved the final manuscript.